Tailoring the expression of Xyr1 leads to efficient production of lignocellulolytic enzymes in Trichoderma reesei for improved saccharification of corncob residues

Background The filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production. Results Here, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC–MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass. Conclusions Our results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-022-02240-9.


Introduction
Lignocellulosic biomass is an abundantly available, inexpensive organic resource on the earth, which is basically composed of cellulose, hemicellulose, and lignin [1]. Biological degradation of lignocellulose biomass by lignocellulolytic enzymes into fermentable sugars for the production of biofuels and other biochemicals has  15:142 received extensive attention [2,3]. However, the high cost of cellulase production, which is the major barrier for practical applications, limits the development of an efficient and economically viable lignocellulosic feedstock based biorefinery [4]. The filamentous fungus Trichoderma reesei encodes a wide repertoire of cellulase, hemicellulase and accessory proteins to degrade cellulose and other biomass components, which makes it be extensively employed in the industrial cellulase production [5]. The cellulases secreted from T. reesei mainly include three enzyme components, namely, cellobiohydrolases (CBHs, EC 3.2.1.91), endoglucanases (EGs, EC3.2.1.4), and β-glucosidase (exactly BGL1, EC 3.2.1.21) [6]. In general, CBHs and EGs act cooperatively to degrade cellulose into cellobiose, and BGL1 ultimately hydrolyzes the oligosaccharide into glucose [6]. The three components work synergistically to accomplish a complete deconstruction of cellulose [7]. Optimizing the composition of cellulase complexes by genetic engineering to strengthen the synergism has been proved to be practical [8,9], among which the overexpression of BGL or EGs is a powerful strategy for enhancing the synergistic effect [10][11][12]. Recently, Qian et al. demonstrated that the double overexpression of EG2 and BGL1 can significantly ameliorate the performance on the cellulase yield and saccharification efficiency in T. reesei [13]. Moreover, it is known that cellulase gene expression is stringently controlled by transcriptional regulatory networks in T. reesei [14]. Therefore, engineering the transcriptional factors can improve the entire cellulase system, which has been considered to be an effective way for enhancing cellulase production.
The energy-efficient production of cellulase in fungi is strictly regulated by a suit of transcription factors [15]. So far, several transcription factors involved in the modulation of cellulase gene expression have been identified, including at least five transcription activators (Xyr1, Ace2, Ace3, Vib1, and the Hap/2/3/5 complex) and four repressors (Cre1, Ace1, Rce1, and Ctf1) [16][17][18][19]. Other transcription factors, such as BglR, Pac1, and Crz1, have also been identified to regulate the biosynthesis of cellulase [20][21][22]. Especially, Xyr1 is the transcriptional activator modulating the expression of cellulase and hemicellulase genes in T. reesei [24]. Overexpression of xyr1 can markedly enhance cellulase production in T. reesei [26,28]. In addition, Xyr1 also seems to be involved in the glucose repression of cellulase mediated by the carbon catabolite repressor Cre1, since its constitutive expression being able to reduce this repressive effect [4,25,26]. It has been reported that the expression levels of the main cellulase genes (cbh1 and cbh2) appeared to be strictly dependent on the expression level of xyr1 [25]. Zheng et al. overexpressed the xyr1 gene under the control of the copper repressible promoter Ptcu1 and the transcript level was improved by approximately twofold on Avicel [27]. However, it seemed that the efficiency of the tcu1 promoter in enhancing transcription of the major cellulase gene cbh1 remains lower compared to the strong pyruvate decarboxylase gene (pdc) promoter, with the transcript level of xyr1 was increased by 16-fold under the similar condition [27,28]. As noted above, tuning the expression dosage of xyr1 may have profound effects on cellulase expression in T. reesei, which requires more systematical and in-depth investigation.
In the present study, the expression of xyr1 was tailored with three differential strong promoters (Pegl2, Pcbh1, and Pcdna1) in T. reesei. We found that the overexpression of xyr1 driven by Pegl2, not that driven by the stronger promoter Pcbh1 or Pcdna1, leads to more significantly enhanced cellulase production. Meanwhile, it also resulted in a significant opening of chromatin in the core promoter regions of cellulase genes, which was accompanied by the activation of the corresponding gene expression. Furthermore, the overexpression of xyr1 with Pegl2 also remarkably improved the levels of accessory proteins involved in lignocellulose degradation and thus promoted the saccharification efficiency toward acidpretreated corncob residues.

Overexpression of the xyr1 gene using the promoters with different strengths in T. reesei
To systematically investigate the effect of differential expression of xyr1 on cellulase production in T. reesei, the xyr1 overexpression cassettes driven by the cellulose-inducible promoters Pegl2, Pcbh1, and the strong constitutive promoter Pcdna1 were constructed and precisely integrated by homologous recombination into the hph locus of T. reesei QEB4, respectively (Fig. 1A). The strains harboring the xyr1 gene driven by the promoters Pegl2, Pcbh1, and Pcdna1 were designated QE2X, QCBX, and QCDX, respectively. The integration of the xyr1 gene into the genome of T. reesei QEB4 was verified by PCR (data not shown). Furthermore, the transcript levels of xyr1 were analyzed by RT-qPCR (Fig. 1B). It was found that xyr1 was significantly overexpressed in the strains, with the maximum transcript levels in the QCDX strain, followed by QCBX and QE2X, which were 47.2-, 12.6-, and 5.8-fold higher than the parental strain QEB4, respectively.
Next, the three xyr1 overexpression strains QE2X, QCBX, and QCDX were cultured on minimal medium (MM) plates containing glucose, glycerol, and lactose as the carbon source, respectively, as well as on PDA plates to investigate the effect of differential xyr1 overexpression on mycelial phenotypes of T. reesei. As shown in Additional file 1: Fig. S1, the colonies of the recombinant strains extended normally and grew at a rate similar to that of the parental strain, indicating that different dosage levels of xyr1 had little effect on mycelial growth of T. reesei. Thereafter, the recombinant strains were inoculated on microcrystalline cellulose (MCC), CMC, and CMCesculin plates to assess the cellulase secretion capacity (Fig. 2). These strains displayed wider hydrolytic haloes or black zones around the colonies relative to the parental strain, suggesting that the cellulase secretion ability was enhanced to different extents. Especially, the cellulase secretion in QE2X strain with the moderately strong promoter Pegl2 was far greater than that in the other two strains with stronger promoters (Pcbh1 and Pcdna1) on the plates, implying that there may be a dose-dependent correlation between the expression level of xyr1 and the secretion of cellulase in T. reesei.

Influences of differential overexpression of xyr1 on the transcriptional profile of cellulase genes
It is known that transcription of the cellulase-encoding genes are modulated by the transcriptional activator Xyr1 [23]. Here, the transcript levels of five main cellulase-encoding genes cbh1, cbh2, egl1, egl2, and bgl1 were measured by RT-qPCR in the xyr1 overexpression strains (Fig. 3A). It was found that the transcript quantities of the five cellulase genes in the QE2X strain with the lowest degree of xyr1 overexpression (5.8-fold) were 7.3-, 5.0-, 1.4-, 4.8-, and 5.8-fold higher than those of the parental strain, respectively. However, those in the QCBX strain with the stronger xyr1 overexpression (12.6-fold) were only slightly increased by 3.2-, 2.5-, 0.9-, 1.2-, and 2.6-fold, respectively. Unexpectedly, the transcript levels of these cellulase genes in the QCDX strain with the highest degree of xyr1 overexpression (47.2-fold) were not significantly improved. Taken together, these results demonstrate that cellulase gene transcription is precisely regulated by the xyr1 transcript abundance, i.e., the moderately strong overexpression of xyr1 with the Pegl2 promoter, but not the stronger overexpression with the Pcbh1 or Pcdna1 promoter, could lead to more pronounced improvement in cellulase expression.
Furthermore, transcript analysis of several transcription factors involved in cellulase expression were performed (Fig. 3B). The positive transcriptional regulators-encoding genes ace2, ace3, and vib1 were virtually unchanged in all the recombinant strains. However, the negative transcriptional regulator-encoding genes cre1, ace1, and ctf1 were greatly decreased in both QE2X and QCBX. As for the QCDX strain, the expression of ace1 and ctf1 showed no significant changes, while the carbon catabolite repressor-encoding gene cre1 was downregulated. These findings suggest that overexpression of xyr1 probably resulted in downregulation of the negative regulators, thus facilitating improvement of the expression of cellulase genes.

Differential overexpression of xyr1 regulated the chromatin accessibility of cellulase genes and the native xyr1 gene
Eukaryotic gene expression is often accompanied by chromatin remodeling [29]. It was reported that the deletion of xyr1 in T. reesei resulted in the loss of expression of the cellulase genes cbh1 and cbh2 and the reduction in chromatin accessibility of these genes [30]. To gain further insight into the impact of xyr1 dosage, the chromatin packing of the core promoter region of cbh1 and cbh2 in the xyr1-overexpressing strains was analyzed by CHART-PCR (Fig. 4A, B). In all the recombinant strains, the chromatin accessibility index (CAI) in the promoter region of cbh1 and cbh2 was improved to varying degree which was corresponded with the high expression levels. This suggests that the xyr1 overexpression can lead to more open chromatin status of cellulase genes. As expected, remarkable chromatin opening of cbh1 and cbh2 was detected in QE2X, indicating that the egl2 promoter-driven xyr1 overexpression is more efficacious in promoting the chromatin accessibility of cellulase-encoding genes than the stronger cbh1/cdna1 promoter-driven overexpression.
It was also known that the expression level of xyr1 may correlate with the degree of chromatin opening in its own promoter [31]. Hence, the chromatin packing of the native xyr1 core promoter region in the xyr1-overexpressing strains was analyzed (Fig. 4C). More open chromatin status was found in all the recombinant strains, with the most significant chromatin opening in QE2X. These results further corroborate the view that the dosage level of xyr1 differentially influenced the chromatin status of its own promoter.

Differential overexpression of xyr1 induced the unfolded protein response in the endoplasmic reticulum
As the major secreted proteins in T. reesei, the newly synthesized cellulase enzymes should enter the endoplasmic reticulum (ER) secretory pathway where they fold into the correct three-dimensional structure with the assistance of resident ER folding factors for secretion, whereas those that misfold would be efficiently degraded by the ER-associated degradation (ERAD) [32]. Meanwhile, the accumulation of unfolded proteins in the ER can cause ER stress, which elicits the unfolded protein response (UPR), a mechanism to upregulate UPR-regulated genes involved in protein folding, ERAD, and others for the restoration of ER homeostasis [33]. To investigate the effect of differential expression of xyr1 on the secretory pathway, the expression of genes encoding key components involved in protein folding (pdi1 and bip1) and the ERAD pathway (hrd1 and der1) was determined in the xyr1-overexpressing strains. As shown in Fig. 5, all tested genes (pdi1, bip1, hrd1, and der1) were significantly upregulated in QE2X, while only the folding factor genes (pdi1 and bip1) were upregulated in QCBX and QCDX. These above results demonstrate that the xyr1 overexpression could induce the UPR in the ER. In particular, the strong ER stress evoked in QE2X resulted in significant activation of the ERAD pathway, indicating a large amount of cellulase has been accumulated in the ER, i.e., saturation of the secretory pathway may be reached in this strain.

Pegl2-driven xyr1 overexpression greatly enhanced cellulase production in T. reesei
To investigate whether the cellulase production levels were improved in the xyr1 overexpression strains, the activities of cellulase in the fermentation broths were determined under Avicel-inducing conditions. The total cellulase activity of QE2X reached 8.4 IU/mL, 3.2-fold higher than that of the parental strain (2.0 IU/mL), using filter paper activity (FPA) as a characterization method, while the FPA in QCBX and QCDX was only elevated 0.9-and 0.8-fold compared to that of the parental strain, respectively (Fig. 6A). Consistently, QE2X also displayed a significant enhancement in the activities of the cellulase components, with 2.1-, 2.4-, and 1.8-fold increase in CBHs, EGs, and BGL activities, respectively. However, QCBX and QCDX only demonstrated 1.4-and 1.3-fold improvement in CBH activity, 1.9-and 1.7-fold improvement in EG activity as well as 1.2-and 1.1-fold In addition, the concentration of extracellular protein in QE2X reached 3.0 mg/mL, much higher than that in QCBX and QCDX (Fig. 6E). Taken together, these results indicate that the egl2 promoter-driven xyr1 overexpression can significantly enhance cellulase production in T. reesei.
Pegl2-driven overexpression of xyr1 optimized the lignocellulolytic enzyme system for saccharification of corncob residues Among the three xyr1 overexpression strains, the Pegl2driven overexpression strain QE2X showed the highest cellulase activity, and hence, it was selected for subsequent saccharification. Two differently pretreated corncob residues, acid-pretreated (ACR) and delignified (DCR) corncob residues, were utilized as substrates to be saccharified with the enzyme preparations produced by QE2X, with the parental strain QEB4 as control.
As shown in Fig. 7, the glucose release from the QE2X enzyme preparation in the saccharification of ACR was 11.9 mg/mL, which was 57.2% higher than that from QEB4. However, no significant difference was observed in the saccharification of DCR between those from QE2X and QEB4. Since the difference in composition between the two corncob substrates is that ACR contains higher levels of lignin than DCR, these results demonstrate that the Pegl2-driven overexpression of xyr1 can optimize the lignocellulolytic enzyme system for saccharification of the high lignin-containing lignocellulosic substrates.
It is known that accessory proteins, such as glucuronoyl esterase (Cip1 and Cip2), swollenin (Swo1), and AA9 lytic polysaccharide monooxygenases (LPMOs), play important roles in enhancing lignocellulose degradation [34]. Especially, Cip1 can reduce the nonproductive adsorption of lignin by cellulase and Cip2 acts in the cleavage of hemicellulose-lignin crosslinks [35,36]. Ma et al. demonstrated by RNA sequencing in T. reesei that Xyr1 could stimulate the expression of those accessory protein-encoding genes [37]. Thus, the transcript levels of the above accessory proteinencoding genes were firstly examined. The transcript levels of cip1 and cip2 in QE2X were improved significantly, which were 21-and 18-fold higher than those in the parental strain QEB4, respectively (Fig. 8). Furthermore, the swollenin-encoding gene swo1 and two LPMO-encoding genes cel61a and cel61b were about 3.4-, 1.3-, and 0.7-fold higher in QE2X than those in QEB4, respectively (Fig. 8). Meanwhile, the LC-MS/ MS analysis of the secretome of T. reesei QE2X and QEB4 was carried out. It was found that, besides the increased amounts of cellulases, the PSMs of all the above-mentioned accessory proteins in QE2X were also higher than those in QEB4 when equal protein was used for evaluation (Table 1), suggesting more accessory proteins were present in the enzyme preparations of QE2X. These results indicate that the Pegl2-driven overexpression of xyr1 can optimize the lignocellulolytic enzyme system of T. reesei by improving the expression of those accessory proteins, which is beneficial for efficient degradation of lignocellulosic biomass.

Discussion
In T. reesei, genetic engineering of transcriptional regulators has been used as a powerful tool for enhancing cellulase production. Xyr1 is the key transcriptional activator of lignocellulolytic genes, whose overexpression can improve cellulase yields under both inducing and non-inducing conditions [26]. However, the effect of xyr1 dosage regulation on cellulase expression has not yet been elucidated. Herein, the expression of xyr1 was tailored under promoters of different strengths and we found that the xyr1 overexpression with the Pegl2 promoter resulted in a substantial improvement in cellulase production and saccharification efficiency.
In previous studies, the promoter Ppdc was chosen to drive xyr1 expression for the construction of cellulase hyperproducing strain [4,28,38]. In addition, the promoter Ptcu1 was also used to the expression of xyr1, and the resultant strain could achieve full cellulase expression [27]. By roughly comparing the effects of the two promoters, we speculate that the overexpression of xyr1 driven by the stronger promoter may have a more pronounced influence on cellulase production. However, this does not imply that the maximization of cellulases can be achieved by extremely strong overexpression of xyr1. Bao et al. found that the moderate overexpression of Sec16 was more potent to increase protein secretion in Saccharomyces cerevisiae than the strong overexpression [39]. Thus, uncertain relationships between the degree of xyr1 overexpression and cellulase gene expression in T. reesei limit the potential for cellulase production to some extent. To further determine whether the effect of xyr1 overexpression depends on its dosage, we selected three strong promoters for tailoring the expression of xyr1: the main cellulase-encoding genes promoters Pegl2 and Pcbh1, as well as the strong constitutive promoter Pcdna1. Among the xyr1 overexpression strains, the strain QE2X with the egl2 promoter exhibited the highest cellulase production, which was consistent with the significantly enhanced cellulase secretion capacity and cellulase gene transcription (Figs. 2, 3A and 6). These findings further suggest that the robust upregulation of cellulase genes caused by the Pegl2-driven overexpression of xyr1 result in the outstanding cellulase production observed in the QE2X strain.
It has been reported that the level of xyr1 transcription tightly regulate the main cellulase genes expression [25]. However, our results showed that the Pegl2-driven xyr1 overexpression (5.8-fold) markedly improved both the transcription and chromatin accessibility of cellulase genes, while the effects of the xyr1 overexpression driven by the cbh1 and cdna1 promoters (12.6-and 47.2-fold) were not particularly remarkable (Figs. 3A, 4A, B). It has been reported that upon cellulase induction, Xyr1 is synthesized and rapidly imports into the nucleus in order to regulate cellulase gene expression [40]. In order to investigate the effect of differential xyr1 overexpression on the Xyr1 protein level, the nuclear protein extracts of the T. reesei strains were also prepared and determined by LC-MS/MS in this study. More peptides of Xyr1 could be detected in QE2X than the other two strains, QCBX and QCDX (Additional file 3: Table S2). That is, the Pegl2driven xyr1 overexpression, instead of the Pcbh1/Pcdna1driven overexpression, significantly improved the amount of the Xyr1 protein in the nucleus. It is known that a phenomenon of "quelling" in filamentous fungi displays the characteristic feature of post-transcriptional gene silencing (PTGS), which functions by mediating mRNA degradation or translational suppression [41,42]. There may be a "quelling" mechanism in T. reesei in which the  excessive mRNA of xyr1 causes translation repression, ultimately leading to a reduction in the amount of Xyr1 in the nucleus. Thus, it can be speculated that the transcript levels of cbh1 and cbh2 do not correspond to the transcript levels of xyr1 to some extent, but strictly to its protein levels.
Meanwhile, Xyr1 has been implicated in chromatin remodeling with histone modification, which has a regulatory role on cellulase gene expression in T. reesei [43]. Mello-de-Sousa et al. have demonstrated that the deletion of xyr1 could trigger chromatin compaction in the upstream regions of the cellulase genes (cbh1 and cbh2) under inducing conditions, which suggests that Xyr1 is necessary for the chromatin opening of these regions [30]. Our results further confirmed this conclusion. The cbh1 and cbh2 promoters of the xyr1-overexpressing strains were more accessible compared to the parental strain (Fig. 4A, B). However, the chromatin packing pattern of cbh1 and cbh2 genes did not exactly follow the transcript level of the xyr1 gene. Among the three strong promoters tested, the moderately strong one, i.e., the Pegl2-driven overexpression of xyr1 was more beneficial for the chromatin accessibility of cellulase-encoding genes, which resulted in the higher gene expression. Furthermore, we also confirmed that Xyr1 could influence the chromatin remodeling of xyr1 itself. The opening of the native xyr1 promoter is more pronounced in the xyr1-overexpressing strains than in the parental strain (Fig. 4C). Till et al. have reported the presence of Xyr1 regulatory element in the xyr1 promoter, which is involved in the Xyr1 autoregulatory mechanism [44]. Thus, the increase of chromatin accessibility of the xyr1 gene may be related to the above mechanism, which warrants further investigation.
BGL is the rate-limiting enzyme in the cellulose hydrolysis process, whose overexpression is beneficial for optimization of cellulase system [12]. However, the overexpression of xyr1 is not adequate to mediate the high-level expression of BGL, which may be attributed to the presence of less Xyr1 binding sites (XBS) on the bgl promoter [4,45]. In the parental strain QEB4, the introduction of bgl gene under control of the strong cbh1 promoter containing more XBS makes the native Xyr1 a limiting step to further improve the expression of bgl [13]. In this study, the overexpression of xyr1 in the strain QEB4 resulted in an increase in the production of BGL by a maximum of 1.8-fold (Fig. 6D). This result indicates that simultaneous engineering of transcription factors and cellulase genes could be a feasible strategy for improvement of cellulase production.
It is known that the efficiency of saccharification of lignocellulosic substrates is closely related to the performance of lignocellulases [11]. However, the composition of the enzyme complexes secreted by T. reesei may not be optimal for industrial applications due to the presence of certain enzymes in limiting amounts [9,46]. Optimization of enzyme complex with different proportions of cellulase components and accessory proteins has been employed for highly efficient bioconversion of lignocellulosic biomass [12,47]. In addition, lignocellulose is embedded in an amorphous matrix of cross-linked lignin and hemicellulose, which limits the cellulase accessibility to substrates [48,49]. Especially, lignin is considered as the rate-limiting factor in the hydrolysis of lignocellulosic biomass as it impedes the access of cellulase to cellulose leading to non-productive binding [50,51]. Hence, delignification of lignocellulosic biomass can improve the saccharification efficiency and saccharide yields [52,53]. BSA and surfactants have also been demonstrated to prevent cellulase-lignin interactions to some extent [54,55]. Furthermore, exogenous addition of Cip1 promoted the degradation of lignocellulosic substrates with high lignin [36]. Analysis of the extracellular proteins of QE2X by LC-MS/MS demonstrated the higher amounts of accessory proteins (Cip1, Cip2, Swo1, and LPMO) in QE2X than the parental strain QEB4 (Table 1), which altered the diversity of enzyme mixtures. Thus, the hydrolysis efficiency of ACR through the crude enzyme produced by QE2X had been greatly improved, showing the Pegl2driven xyr1 overexpression could optimize the lignocellulolytic enzyme system by improving expression of accessory proteins.

Conclusion
In the present study, we showed that the Pegl2-driven xyr1 overexpression significantly improved production of lignocellulolytic enzymes in T. reesei (Fig. 9). The total cellulase activity of QE2X was up to 8.4 IU/mL under inducing condition. The glucose release from ACR by the enzyme mixture of QE2X was improved by 57.2%. To the best of our knowledge, this is the first report regarding the roles of xyr1 dosage level on cellulase expression in T. reesei. The significantly enhanced production of cellulase in QE2X is attributed to the moderate upregulation of transcription factor, with the underlying mechanisms require further exploration. In conclusion, this study demonstrates that selecting the promoter with proper dosage to drive the expression of key regulators is a feasible strategy to enhance lignocellulolytic enzyme production for the conversion of lignocellulose biomass.

Strains, medium, and culture conditions
T. reesei QEB4, an EG2-BGL1 double overexpression strain [13], was used as the parental strain in this study. The strain QEB4 was constructed from the uracil auxotrophic strain QP4 whose pyr4 gene was replaced with the resistance gene hph. The fungal strain and its derivatives were cultivated on potato dextrose agar (PDA) plates at 30 ℃ for 5-7 days to produce conidia, which were harvested, and then inoculated with approximately 10 6 spores/mL into 50 mL of liquid minimal medium (MM) [56] before incubation for 36 h, 30 ℃ and 200 rpm. Then, equal amounts (0.4 g cell wet weight) of mycelia were transferred into 100 mL of the cellulase production medium (CPM) in 500 mL Erlenmeyer flasks. The CPM solution was composed as follows: 2% (w/v) Avicel, 0.1% CaCl 2 ·2H 2 O, 0.5% (NH 4 ) 2 SO 4 , 0.5% KH 2 PO 4 , 0.06% MgSO 4 ·7H 2 O, and 2% corn steep liquor.

Construction of the xyr1 overexpression strains
The xyr1 overexpression cassettes were constructed with the double-joint PCR method [57]. The Phanta ® Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd., Nanjing, China) was used for PCR amplification, and DNA fragments were purified using Gel Extraction Kit (Omega, USA). The primers were designed using the primer premier 5.0 software. Primer synthesis and DNA sequencing were performed at BGI Genomics (Qingdao, China). The primers used in this study are presented in Additional file 2: Table S1. The plasmid T-ptrA was used as the template to clone the ptrA expression cassette by PtrA-F/PtrA-R. The 5′-and 3′-flank fragments of hph, the three promoters (egl2, cbh1, and cdna1), and the encoding region and terminator of xyr1 were amplified from the genomic DNA of T. reesei QEB4 using the primer pairs Hph-UF/Hph-UR, Hph-DF/Hph-DR, Egl2-F/Egl2-R, Cbh1-F/Cbh1-R, Cdna1-F/Cdna1-R, and Xyr1-F/Xyr1-R, respectively. Subsequently, the purified DNA fragments were fused together in 1:2:4:2:1 molar ratio of 5′-flanking region:ptrA: promoter:xyr1:3′-flanking region and were used as the template to amplify the final expression cassettes by Hph-cUF/Hph-cDR. The cassettes were transformed into the protoplasts of T. reesei QEB4 using the method described previously, respectively [56].
The strains were screened on MM agar plates containing 300 μg/mL pyrithiamine, and the purified candidate transformants were identified and named as QE2X, QCBX, and QCDX, respectively.

RNA extraction and RT-qPCR analysis
The fungal mycelia were collected and flash-frozen in liquid nitrogen. Total RNA was extracted using the Trizol reagent (TaKaRa, Japan), and the RNA concentration was quantified by using a NanoDrop LiTE Spectrophotometer (THERMO SCIENTIFIC, United States). 1 μg RNA was reverse transcribed to cDNA using PrimeScript RT reagent Kit (TaKaRa, Japan) following the manufacturer's protocol. RT-qPCR analysis was performed with SYBR Premix Ex Taq ™ (TaKaRa, Japan) and the LigntCycler 480 system (Roche, Mannheim, Germany) using the primers listed in Additional file 2: Table S1. Three biological replicates were carried out for each sample. Transcript levels of target genes were analyzed according to the ΔΔCt method using the actin gene for normalization.

Growth and secretion ability analysis
To analyze the growth morphology on plate, the parental and recombinant strains were pre-grown on MM agar plates and then equal amounts of mycelia were transferred in biological duplicates onto PDA plates or MM plates containing 2% glucose, 2% glycerol, or 2% lactose, respectively, and incubated at 30 ℃ for 2 days. Mycelia diameters were measured every 12 h for 2 days and recorded as representation of growth rates.
To determine the secretion ability of total cellulase, the equal amount of mycelia was inoculated on MCC plate containing 0.05% ball-milled Avicel, 0.02% peptone on the upper layer, and water agar on the bottom layer. After 5 days incubation, the double-layer MCC plate with hydrolysis zones was measured and recorded by photographs. In addition, the secretion ability of EGs and BGL1 in recombinant strains was assayed by the CMCesculin plates (1% CMC-Na, 0.3% esculin, 0.05% ferric citrate and 2% agar) and the CMC plates (1% CMC-Na, 0.1% yeast extract and 2% agar), respectively.

Enzyme activity and protein assay
The activities of FPase (FPA) and EGs were determined by measuring the released reducing sugar with filter paper and CMC-Na as substrates, respectively. Determination of FPA was performed in a 2 mL reaction system containing 500 μL of the appropriately diluted culture supernatant, 1.5 mL of 50 mM citrate buffer (pH 4.8), and 0.05 g of Whatman No.1 filter paper. The EGs activity assay was carried out at 50 ℃ in a 2 mL reaction system including 500 μL of the suitably diluted supernatant and 1.5 mL of 0.5% CMC sodium in 50 mM citrate buffer (PH 4.8). The DNS method was used to measure the amount of released reducing sugars [58]. The CBHs and BGL activities were determined by measuring the amount of released p-nitrophenol using p-nitrophenyl-D-cellobioside (pNPC, Sigma, USA) and p-nitrophenyl-β-D-glucopyranoside (pNPG, Sigma, USA) as the substrates, respectively. The assays were performed in 200 μL reaction mixtures containing 50 μL of the diluted supernatant, 50 μL of the respective substrate, and 100 μL of 50 mM citrate buffer (pH 4.8) and then incubated at 50 ℃ for 30 min. One unit (U) of enzyme activity was defined as the amount of enzymes liberating 1 µmoL reducing sugars (FPA, EGs) or p-nitrophenol (CBHs, BGL1) per minute under the test conditions. Total extracellular proteins were assayed as previously described by Ellilä et al. [4]. Briefly, proteins were precipitated with ice-cold acetone and the concentration was quantified using the Bradford Kit (Sangon Biotech, Shanghai, China) with bovine serum albumin (BSA) as a standard. To facilitate analyzing the amount of the Xyr1 protein, total nuclear protein extracts were prepared from the T. reesei strains cultured on 2% (w/v) Avicel for 3 days. And the extraction experiment was performed using the Filamentous fungi Nuclear Protein Extraction Kit (BestBio, Shanghai, China) according to the manufacturer's instructions.

Southern blot analysis
The specific probe was amplified from the genome of T. reesei QEB4 using the primer pair Hph-pF/Hph-pR. Chromosomal DNA of the parental and recombinant strains was digested thoroughly by Mph1103I and then hybridized by the probe. Finally, the probe-hybridized DNA fragments were detected and visualized using a DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Germany) according to the manufacturer's instructions.

Chromatin accessibility real-time PCR (CHART-PCR) assays
CHART-PCR assay were performed according to a previously described protocol [60]. Briefly, mycelia were collected after 48 h of induction, filtered, and ground in liquid nitrogen. Subsequently, 0.1 g powder was incubated with 10 μL RNase-free DNase I (TaKaRa Biotechnology) for 5 min at 37 ℃ in 1 mL of nuclease digestion buffer (250 mM sucrose, 0.05 mM CaCl 2 , 3 mM MgCl 2 , 15 mM NaCl, 60 mM KCl, 5 mM DTT, and 15 mM pH 7.5 Tris-HCl). The reaction was terminated by adding 100 μL of termination buffer (20 mM EDTA and 2% SDS). Then, two rounds of phenol-chloroform extraction and one round of chloroform extraction were followed for protein extraction. The supernatant was treated with RNase A (10 μg/mL) at 37 ℃ for 15 min and precipitated with 0.3 M NaAc and two volumes of ethanol. The DNA obtained were dissolved in 30 μL of double-distilled water. A negative control reaction without DNase I was included. Quantitative PCR analysis on the DNase I-treatment samples was performed to measure the relative abundance of target regions. Each sample was prepared in triplicate. The chromatin accessibility index (CAI) was defined as CAI = Dc/Ds, where Dc is the amount of intact DNA detected for the promoter regions of actin gene and Ds is the amount of intact DNA detected for each target region. The amount of intact input DNA for each sample was calculated by comparing the thresholds of the PCR amplification plots with the standard curve produced for each primer set using serial dilutions of undigested genomic DNA. All primer sequences are provided in Additional file 2: Table S1.

LC-MS/MS
LC-MS/MS analyses were performed according to the method described previously with appropriate modifications [61]. The fermentation supernatants of three biological replicates were mixed in equal proportions and then subjected to the measurement. The sample (100 μg of protein, measured by the Bradford method) was denatured in an equal volume of 8 M urea and reduced in 5 mM DTT at 56 ℃ for 30 min. The samples were then alkylated in 14 mM iodoacetamide (IAA) for 1 h in darkness. The unreacted IAA was quenched by the addition of 5 mM DTT for 15 min. The samples were digested by adding trypsin (Sigma, USA) with 1 mM CaCl 2 as a co-factor at 37 ℃ for 12 h, and reaction was terminated by adjusting to pH 2 with trifluoroacetic acid. The digested oligopeptides were desalted on C18 Sep-Pak cartridges (Waters Associates, USA) and were further dissolved in 0.1% formic acid and then were subjected to nanoelectrospray ionization, followed by analysis in LTQ Orbitrap Velos Pro (Thermo Scientific, USA) coupled with HPLC system. The Orbitrap resolution was set at 70,000, and data from LC-MS/MS analysis were searched by proteome discovered software 1.4 (Thermo Fisher Scientific). Sequences were mapped based on the reference genome of T. reesei, acquired from the JGI Genome Portal (https:// mycoc osm. jgi. doe. gov/ Trire2/ Trire2. home. html). Furthermore, the relative protein abundance was characterized by peptide spectrum matches (PSMs), which could correlate linearly with the protein abundance [62].